(Adapted from the applicant's abstract): Vacuolar H+ATPases (V-ATPases) have an essential role in renal bicarbonate transport in several segments of the nephron. V-ATPases also reside in intracellular membrane compartments of all eukaryotic cells, serving to acidify the vacuolar system. The plasma membrane V-ATPases of H+-transporting cells have several features that distinguish them from V-ATPases of intracellular organelles: 1) they reside at high densities on the plasma membrane, 2) their amplified expression occurs in a cell-specific manner; 3) they have a polarized distribution that allows for vectorial secretion of hydrogen ion across the epithelial layer; and 4) the plasma membrane V-ATPases of the nephron are subject to physiologic regulation, enabling the kidney to preserve acid base balance. Despite the widespread physiologic importance of plasma membrane V-ATPases, the cellular mechanisms underlying their amplification, traffic to the plasma membrane, and regulation remain poorly understood. Two major impediments are the lack of cultured cell lines that maintain high levels of V-ATPase on the plasma membrane, and the lack of tools to study the biochemistry and cell biology of V-ATPases residing on the plasma membrane. The overall goal of this application is to advance our understanding of the distinctive properties of plasma membrane V-ATPases in renal epithelial cells, and top develop new approaches for studying them. The specific aims are: 1. Investigate cellular mechanisms controlling V-ATPase amplification and targeting to the plasma membrane. a. Determine whether overexpression of mRNA for the V-ATPase B1 and E subunits affects cellular V-ATPase abundance, assembly, and distribution, and whether it affects the expression of other V- ATPase subunits. b. Examine the role of translational control and subunit degradation in the regulation of V-ATPase subunit protein abundance. c. Develop antibodies that recognize extracellular determinants on a functional vacuolar H+ATPase. 2. Investigate the role of V-ATPase assembly/disassembly in renal bicarbonate secretion. a. Determine whether lowering extracellular pH induces basolateral membrane V-ATPase disassembly in beta intercalated cells in vitro. b. Determine whether lowering extracellular pH induces plasma membrane V-ATPase disassembly in cultured renal epithelial cells. 3. Develop an immortalized intercalated cell line using B1 subunit promoter-driven transforming antigen constructs in transgenic mice.